The primary focus of the section is to further our understanding of the molecular basis of signaling between G protein coupled receptors and voltage gated ion channels in neurons using electrophysiological, molecular, and imaging techniques. There are four main projects currently underway. The effects of RGK family small GTPase proteins on N-type calcium channels in sympathetic neurons. Rad, Gem/Kir, Rem and Rem2 are members of the Ras-related RGK family of small GTP-binding proteins. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels requires both the Ras homology (core) domain and the polybasic C-terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 does not affect suppression of calcium currents. Loading neurons with GDP-beta-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, radiolabeled conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Taken together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel beta-subunits in the preassembled N-type channel thereby forming a non-conducting species. Future studies aimed at determining the precise molecular mechanism and physiological roles of Rem2 are planned. Group III metabotropic glutamate receptors (mGluRs; mGluR4, 6, 7, and 8) coupling to voltage-gated calcium channels is being examined. Although most mGluRs have been functionally expressed in a variety of systems, few studies have demonstrated robust coupling of mGluR8 to downstream effectors. We therefore tested whether activation of mGluR8 inhibited calcium channels. Both L-glutamate (L-Glu) and L-AP4, a selective agonist for group III mGluRs, inhibited N-type Ca2+ current in rat SCG neurons previously injected with a cDNA encoding mGluR8a/b. The potency and efficacy of L-AP4 and L-Glu were similar for both splice variants. Agonist-induced inhibition was abolished by pretreatment with CPPG, a selective group III mGluR antagonist, and Pertussis toxin. Deletion of either a calmodulin (CaM) binding motif in the C-terminus or the entire C-terminus of mGluR8 did not affect mGluR8-mediated response. Our studies indicate that both mGluR8a and 8b are capable of inhibiting N-type calcium channel, suggesting a role as presynaptic autoreceptors to regulate neuronal excitability. The studies also imply that the potential CaM binding domain is not required for the mGluR8-mediated calcium channel inhibition and the C-terminus of mGluR8a is dispensable for receptor coupling to N-type Ca2+ channels. Extension of these findings using siRNA and FALI (fluorophore-assisted light inactivation) technologies are underway. Phosducin (PDC) and phosducin-like protein (PDCL) bind to G-protein beta/gamma subunits and disrupt signaling between GPCRs and effectors. The latter protein has also been shown to be up-regulated in neuroblastoma cells treated with ethanol. We have found that expressing PDC and PDCL in dissociated superior cervical ganglion (SCG) neurons attenuates voltage-dependent inhibition of N-type calcium channels in a time-dependent manner. These data are consistent with a model whereby PDC and PDCL physically remove liberated G-protein beta/gamma from the plasma membrane. We are also attempting to develop a fluorescent biosensor based on these molecules that will detect the initial step of G-protein activation, i.e., the release of the Gbeta/gamma subunit. The current strategy involves tagging phosducin or phosducin-like protein at both termini with fluorescent proteins. Binding of G-protein beta/gamma subunits should bring the termini of the proteins into apposition thus allowing FRET to be used as a method of detection. The principle motivation behind these studies is to develop a system that would allow universal high throughput detection of ligands and GPCRs, subcellular localization of GPCR activation, and real-time kinetic analysis of G-protein activation. NaV1.8 is tetrodotoxin-resistant voltage-gated sodium channel that is normally expressed in a subset of sensory neurons involved in pain transmission, but also appears ectopically in neurons damaged by the progression of multiple sclerosis. The genomic basis for normal and abnormal expression is contained within the Scn10a gene encoding the NaV1.8. We have located and isolated the tissue specific promoter for this gene, and have begun dissecting regions conferring both sensory neuron and general neuron specificity. This promoter may be a valuable tool for studying nociceptors, and pain transmission, and may provide information allowing for the design and/or delivery of analgesics directly and specifically to nociceptors.